The present invention relates to methods of converting a maltogenic alpha-amylase into a cyclodextrin glucanotransferase (CGTase) or vice versa or creating hybrids of the two. The invention also relates to the variants made by the methods.
Cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19) and maltogenic alpha-amylase (EC 3.2.1.133) are two classes of glycosylases that degrade starch by hydrolysis of the xcex1-(1,4)-glycosidic bonds, but the initial products are predominantly cyclic for CGTases and linear for the maltogenic alpha-amylase.
Cyclomaltodextrin glucanotransferase (E.C. 2.4.1.19), also designated cyclodextrin glucanotransferase or cyclodextrin glycosyltransferase, abbreviated herein as CGTase, catalyses the conversion of starch and similar substrates into cyclomaltodextrins via an intramolecular transglycosylation reaction, thereby forming cyclomaltodextrins (or CD) of various sizes. Commercially most important are cyclodextrins of 6, 7 and 8 glucose units, termed xcex1-, xcex2- and xcex3-cyclodextrins, respectively.
CGTases are widely distributed and from several different bacterial sources, including Bacillus, Brevibacterium, Clostridium, Corynebacterium, Klebsiella, Micrococcus, Thermoanaerobacter and Thermoanaerobacterium have been extensively described in the literature. A CGTase produced by Thermoanaerobacter sp. has been reported in Norman B E, Jxc3x8rgensen S T; Denpun Kagaku 1992 39 99-106, and WO 89/03421, and the amino acid sequence has been disclosed in WO 96/33267. The sequence of CGTases from Thermoanaerobacterium thermosulfurigenes and from Bacillus circulansis available on the Internet (SCOP or PDF home pages) as pdf file 1CIU, and the sequence of a CGTase from B. circulans is available as pdf file 1CDG.
Tachibana, Y., Journal of Fermentation and Bioengineering, 83 (6), 540-548 (1997) describes the cloning and expression of a CGTase. Variants of CGTases have been described by Kim, Y. H., Biochemistry and Molecular Biology International, 41 (2), 227-234 (1997); Sin K-A, Journal of Biotechnology, 32 (3), 283-288 (1994); D Penninga, Biochemistry, 34 (10), 3368-3376 (1995); and WO 96/33267.
Maltogenic alpha-amylase (glucan 1,4-a-maltohydrolase, E.C. 3.2.1.133) is able to hydrolyze amylose and amylopectin to maltose in the alpha-configuration, and is also able to hydrolyze maltotriose as well as cyclodextrin.
A maltogenic alpha-amylase from Bacillus (EP 120 693) is commercially available under the trade name Novamyl(copyright) (product of Novo Nordisk A/S, Denmark) and is widely used in the baking industry as an anti-staling agent due to its ability to reduce retrogradation of starch (WO 91/04669).
The maltogenic alpha-amylase Novamyl(copyright) shares several characteristics with cyclodextrin glucanotransferases (CGTases), including sequence homology (Henrissat B, Bairoch A; Biochem. J., 316, 695-696 (1996)) and formation of transglycosylation products (Christophersen, C., et al., 1997, Starch, vol. 50, No. 1, 39-45).
The inventors have discovered some striking, and not previously predicted structural similarities and differences between the structure of Novamyl and the reported structures of CGTases, and based on this they have constructed variants of maltogenic alpha-amylase having CGTase activity and variants of CGTase having maltogenic alpha-amylase activity. Further, on the basis of sequence homology between Novamyl(copyright) and CGTases, the inventors have constructed hybrid enzymes with one or more improvements to specific properties of the parent enzymes, using recombinant DNA methodology.
Accordingly, the present invention provides a polypeptide which:
a) has at least 70% identity to amino acids 1-686 of SEQ ID NO: 1;
b) comprises an amino acid modification which is an insertion, substitution or deletion compared to SEQ ID NO: 1 in a region corresponding to amino acids 40-43, 78-85, 136-139, 173-180, 188-195 or 259-268; and
c) has the ability to form cyclodextrin when acting on starch.
The invention also provides a polypeptide which:
a) has an amino acid sequence having at least 70% identity to a parent cyclodextrin glucanotransferase (CGTase);
b) comprises an amino acid modification which is an insertion, substitution or deletion compared to the parent CGTase in a region corresponding to amino acids 40-43, 78-85, 136-139, 173-180, 188-195 or 259-268 of SEQ ID NO: 1; and
c) has the ability to form linear oligosaccharides when acting on starch.
Further, the invention provides a method for constructing a maltogenic alpha-amylase, comprising:
a) recombining DNA encoding a cyclodextrin glucanotransferase (CGTase) and DNA encoding a maltogenic alpha-amylase;
b) using the recombinant DNA to express a polypeptide; and
c) testing the polypeptide to select a polypeptide having the ability to form linear oligosaccharides when acting on starch.
Finally, the invention provides a method of selecting DNA encoding maltogenic alpha-amylase in a DNA pool, comprising:
a) amplifying DNA encoding maltogenic alpha-amylase by a polymerase chain reaction (PCR) using primers encoding a partial amino acid sequence of amino acids 1-686 of SEQ ID NO: 1, preferably comprising at least 5 amino acid residues, preferably comprising one or more of positions 188-196, more preferably comprising positions 190-194,
b) cloning and expressing the amplified DNA, and
c) screening for maltogenic alpha-amylase activity.